This invention relates to a series of ketolide antibacterials in the macrolide family, intermediates used in their manufacture and pharmaceutical compositions containing them. The compounds are erythromycin analogues useful in the treatment of bacterial and protozoal infections and in the treatment of other conditions involving gastric motility.
Polyketides are a family of natural products that include many compounds possessing antibiotic and other pharmacologic properties. Erythromycins are a class of macrolide antibiotics originally discovered in 1952 in the metabolic products of a strain of Streptomyces erythreus. The antibiotic occurs in various glycosylated forms, designated A, B, C, and D. Since their discovery, many have worked to prepare derivatives of the molecule to improve or modify its properties. The focus of much of this work involved chemical modification of the naturally produced erythromycin molecule. For example, clarithromycin is a semi-synthetic antibiotic that is made by chemically modifying the hydroxyl group at C-6 to xe2x80x94OMe.
Ketolides are erythromycin derivatives where the C-3 cladinose sugar is chemically removed and the resulting free hydroxyl group converted into a keto group. For example, U.S. Pat. No. 6,124,269 describes ketolides with a cyclic carbamate group at C-11 and C-12 and an O-alkylaryl group at C-6. U.S. Pat. No. 5,635,485 also describes ketolides with a cyclic carbamate group at C-11 and C-12 but which have a xe2x80x94OMe group at C-6 and an alkylaryl group at the carbamate nitrogen. However, because of the complexity of the macrolide molecule, medicinal chemistry efforts to produce derivatives have been limited by the kinds of modifications that can be made to the naturally occurring erythromycins and their precursors.
Recently, the discovery and isolation of modular polyketide synthases (xe2x80x9cPKS""sxe2x80x9d) have expanded the scope of macrolide structures that may be made. PKS""s are multifunctional enzymes related to fatty acid synthases, which catalyze the formation of the polyketide chains through repeated reactions between its acylthioesters.
The S. erythraea PKS is an assembly of three multifunctional proteins encoded by three separate genes and is described by U.S. Pat. Nos. 5,824,513, 6,004,787, 6,060,234, and 6,063,561. The S. erythraea PKS product is 6-deoxyerythronolide B which is subsequently processed by additional tailoring enzymes to make erythromycins A-D. The collective assembly of the PKS gene and the genes for the tailoring enzymes are referred to as the biosynthetic gene cluster. The S erythraea PKS biosynthetic gene cluster is described by Donadio et al. in Industrial Microorganisms: Basic and Applied Molecular Genetics, (1993), R. H. Balz, G. D. Hegeman, and P. L. Skatrud (eds.), Amer. Soc. Microbiol.
Recombinant methods using vectors encoding a variety of PKS""s, including the PKS from S. erythraea, to make novel polyketides are described by U.S. Pat. Nos. 5,672,491, 5,830,750, 5,672,491, 5,712,146, 5,962,290, 6,022,731, 6,066,721, and 6,077,696. PCT Publication No. WO 98/01546 describes additional methods for modifying the loading domain and thus varying the nature of the starter units that initiate polyketide synthesis. Methods for making polyketides in a cell-free system are described, for example by U.S. Pat. No. 6,080,555 and PCT Publication No. WO 97/02358. Using these techniques, erythromycin analogues where the naturally occurring ethyl group at C-13 is replaced with other groups have been reported, for example in PCT Publication Nos.: WO 97/23630; WO 98/01571, WO 99/35157, WO 00/03986, and WO 00/44761.
Due to the alarming increase in the incidence of resistant strains to currently used antibiotics, a need exists for novel compounds having antibiotic activity, particularly against resistant strains. The present invention fulfills this need by providing novel erythromycin derivatives. These compounds are generally the product of semi-synthesis or the chemical modification of unnatural erythromycin analogues that result from the manipulation of PKS gene clusters.
The present invention relates to novel compounds that are expected to possess antibacterial activity against a broad-spectrum of bacterial strains and are thus useful for the treatment of bacterial infections in humans and animals. The present invention is concerned with compounds of the formula: 
wherein:
X is hydrogen or halide;
R2 is hydrogen, acyl, or a hydroxy protecting group;
R6 is hydrogen, hydroxyl, or xe2x80x94ORa wherein Ra is a substituted or unsubstituted moiety selected from the group consisting of C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, heterocyclo, aryl(C1-C10)alkyl, aryl(C2-C10)alkenyl, aryl(C2-C10)alkynyl, heterocyclo(C1-C10)alkyl, heterocyclo(C2-C10)alkenyl, and heterocyclo(C2-C10)alkynyl;
R13 is hydrogen or a substituted or unsubstituted moiety wherein the moiety is selected from the group consisting of methyl; C3-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, heterocyclo, aryl(C1-C10)alkyl, aryl(C2-C10)alkenyl, aryl(C2-C10)alkynyl, heterocyclo(C1-C10)alkyl, heterocyclo(C2-C10)alkenyl, and heterocyclo(C2-C10)alkynyl;
and,
R is hydrogen or a substituted or unsubstituted moiety wherein the moiety is selected from the group consisting of C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, heterocyclo, aryl(C1-C10)alkyl, aryl(C2-C10)alkenyl, aryl(C2-C10)alkynyl, heterocyclo(C1-C10)alkyl, heterocyclo(C2-C10)alkenyl, and heterocyclo(C2-C10)alkynyl;
and the pharmaceutically acceptable salts, esters and pro-drug forms thereof.
The present invention relates to novel erythromycin derivatives and intermediates thereto. In general, the inventive compounds possess antibacterial activity against Gram positive, Gram negative, and anaerobic bacteria, and are useful as broad-spectrum antibacterial agents for the treatment of bacterial infections in humans and animals. These compounds are effective against diverse strains including but not limited to S. aureus, S. epidermidis, S. pneumoniae, S. pyogenes, enterococci, Moraxella catarrhalis and H. influenzae. Exemplary infections that may be treated include community-acquired pneumonia, upper and lower respiratory tract infections, skin and soft tissue infections, meningitis, hospital-acquired long infections, and bone and joint infections.
Many of the inventive compounds contain one or more chiral centers. All of the stereoisomers are included within the scope of the invention, as pure compounds as well as mixtures of stereoisomers. Similarly, all geometric isomers are also included within the scope of the invention. Where the compounds according to this invention have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
For use in medicine, the salts of the compounds of this invention refer to non-toxic xe2x80x9cpharmaceutically acceptable salts.xe2x80x9d Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate.
The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term xe2x80x9cadministeringxe2x80x9d shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d, ed. H. Bundgaard, Elsevier, 1985.
Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
When a particular group is xe2x80x9csubstitutedxe2x80x9d (e.g., cycloalkyl, aryl, heterocyclyl, heteroaryl), that group may have one or more substituents, preferably from one to five substituents, more preferably from one to three substituents, most preferably from one to two substituents, independently selected from the list of substituents. It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein. Examples of suitable substituents include alkyl, alkenyl, alkynyl, aryl, halo, trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, alkanoyl, alkanoyloxy, amino, alkylamino, aralkylamino, cycloalkylamino, heterocycloamino, dialkylamino, alkanoylamino, thio, alkylthio, cycloalkylthio, heterocyclothio, ureido, nitro, cyano, carboxy, caroboxylalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, alkylsulfonyl, sulfonamindo, aryloxy, and the like, in addition to those otherwise specified herein. The substituent may be further substituted, for example, by halo, hydroxy, alkyl, alkoxy; aryl, substituted aryl, substituted alkyl, substituted aralkyl, and the like.
Under standard nomenclature used throughout this disclosure, the terminal portion of the designated side chain is described first, followed by the adjacent functionality toward the point of attachment. Thus, for example, a xe2x80x9cphenyl(alkyl)amido(alkyl)xe2x80x9d substituent refers to a group of the formula 
The term xe2x80x9csubjectxe2x80x9d as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, the term xe2x80x9ccompositionxe2x80x9d is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
The term xe2x80x9calkylxe2x80x9d refers to straight or branched chain hydrocarbons. xe2x80x9cAlkenylxe2x80x9d refers to a straight or branched chain hydrocarbon with at least one carbon-carbon double bond. xe2x80x9cAlkynylxe2x80x9d refers to a straight or branched chain hydrocarbon with at least one carbon-carbon triple bound.
The terms xe2x80x9csubstituted alkyl,xe2x80x9d xe2x80x9csubstituted alkenyl,xe2x80x9d or xe2x80x9csubstituted alkynylxe2x80x9d refer to the respective alkyl, alkenyl or alkynyl group substituted by one or more substituents. Illustrative examples of substituents include but are not limited to alkyl, alkenyl, alkynyl, aryl, halo; trifluoromethyl; trifluoromethoxy; hydroxy; alkoxy; cycloalkoxy; heterocyclooxy; oxo; alkanoyl (xe2x80x94C(xe2x95x90O)-alkyl); aryloxy; alkanoyloxy; amino; alkylamino; arylamino; aralkylamino; cycloalkylamino; heterocycloamino; disubstituted amines in which the two amino substituents are selected from alkyl, aryl, or aralkyl; alkanoylamino; aroylamino; aralkanoylamino; substituted alkanoylamino; substituted arylamino; substituted aralkanoylamino; thiol; alkylthio; arylthio; aralkylthio; cycloalkylthio; heterocyclothio; alkylthiono; arylthiono; aralkylthiono; alkylsulfonyl; arylsulfonyl; aralkylsulfonyl; sulfonamido (e.g., SO2NH2); substituted sulfonamido; nitro; cyano; carboxy; carbamyl (e.g., CONH2); substituted carbamyl (e.g., xe2x80x94C(xe2x95x90O)NRRxe2x80x2 where R and Rxe2x80x2 are each independently hydrogen, alkyl, aryl, aralkyl and the like); alkoxycarbonyl, aryl, substituted aryl, guanidino, and heterocyclo such as indoyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where applicable, the substituent may be further substituted such as with halogen, alkyl, alkoxy, aryl, or aralkyl and the like.
The term xe2x80x9cacylxe2x80x9d refers to an Rxe2x80x94COxe2x80x94 group wherein R is an alkyl group, typically a C1-C6 lower alkyl group.
The terms xe2x80x9chalogen,xe2x80x9d xe2x80x9chaloxe2x80x9d, or xe2x80x9chalidexe2x80x9d refer to fluorine, chlorine, bromine and iodine.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as phenyl, napthyl, and biphenyl and the like, each of which may be substituted.
The terms xe2x80x9calkylarylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d refer to an aryl group bonded directly through an alkyl group, such as benzyl. Similarly, xe2x80x9carylalkenylxe2x80x9d and xe2x80x9carylalkynylxe2x80x9d refer to an aryl group bonded directly through an alkenyl or alkynyl group respectively.
The term xe2x80x9csubstituted arylxe2x80x9d refers to an aryl group substituted by, for example, one to four substituents such as substituted and unsubstituted alkyl, alkenyl, alkynyl, and aryl; halo; trifluoromethoxy; trifluoromethyl; hydroxy; alkoxy; cycloalkyloxy; heterocyclooxy; alkanoyl; alkanoyloxy; amino; alkylamino; aralkylamino; cycloalkylamino; heterocycloamino; dialkylamino; alkanoylamino; thio; alkylthio; cycloalkylthio; heterocyclothio; ureido; nitro; cyano; carboxy; carboxyalkyl; carbamyl; alkoxycarbonyl; alkylthiono; arylthiono; alkylsulfonyl; sulfonamido; aryloxy; and the like. The substituent may be further substituted, for example, by halo, hydroxy; alkyl, alkoxy; aryl, substituted aryl, substituted alkyl, substituted aralkyl, and the like.
The term xe2x80x9ccycloalkylxe2x80x9d refers to optionally substituted, saturated cyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring which may be further fused with an unsaturated C3-C7 carbocyclic ring. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl; and adamantyl. Exemplary substituents include one or more alkyl groups or one or more groups described above as alkyl substituents.
The terms xe2x80x9cheterocycle,xe2x80x9d xe2x80x9cheterocyclic,xe2x80x9d and xe2x80x9cheterocycloxe2x80x9d refer to an optionally substituted, fully saturated or unsaturated, aromatic or nonaromatic cyclic group, for example, which is a 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring system, which has at least one heteroatom in at least one carbon atom containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, or 3 heteroatoms selected from nitrogen atoms, oxygen atoms, and sulfur atoms, where the nitrogen and sulfur heteroatoms may also optionally be oxidized. The nitrogen atoms may optionally be quatemized. The heterocyclic group may be attached at any heteroatom or carbon atom.
Exemplary monocyclic heterocyclic groups include pyrrolidinyl; pyrrolyl; indolyl; pyrazolyl; oxetanyl; pyrazolinyl; imidazofyl; imidazolinyl; imidazolidinyl; oxazolyl; oxazolidinyl; isoxazolinyl; isoxazolyl; thiazolyl; thiadazolyl; thiazolidinyl; isothiazolyl; isothiazolidinyl; furyl; tetrahydrofuryl; thienyl; oxadiazolyl; piperidinyl; piperazinyl; 2-oxopiperazinyl; 2-oxopiperidinyl; 2-oxopyrrolidinyl; 2-oxazepinyl; azepinyl; 4-piperidonyl; pyridinyl; N-oxo-pyridyl; pyrazinyl; pyrimidinyl; pyridazinyl; tetrahydropyranyl; tetrahydrothiopyranyl; tetrahydrothiopyranyl sulfone; morpholinyl; thiomorpholinyl; thiomorpholinyl sulfoxide; thiomorpholinyl sulfone; 1,3-dioxolane; 1-dioxothienyl; dioxanyl; thientanyl; thiiranyl; triazinyl; triazolyl and the like. Preferred heterocyclo groups include pyridinyl; pyrazinyl; pyrimidinyl; pyrrolyl; pyrazolyl; imidazolyl; thiazolyl; oxazolyl; isoxazolyl; thiadiazolyl; oxadiazolyl; thienyl; furanyl; quinolinyl; isoquinolinyl, and the like.
Exemplary bicyclic heterocyclic groups include benzothiazolyl; benzoxazolyl; benzothienyl; quinuclidinyl; quinolinyl; quinolinyl-N-oxide; tetrahydroisoquinolinyl; isoquinolinyl; benzimidazolyl; benzopyranyl; indolizinyl; benzofuryl; chromonyl; coumarinyl; cinnolinyl; quinoxalinyl; indazolyl; pyrrolopyridinyl; furopyridinyl (such as furo[2,3-c]pyridinyl, fuiro[3,2-b]pyridinyl, or furo[2,3-b]pyridinyl); imidazopyridinyl (such as imidazo[4,5-b]pyridinyl or imidazo[4,5-c]pyridinyl); dihydroisoindolyl; dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl); benzisothiazolyl; benzisoxazolyl; benzodiazinyl; benzofurazanyl; benzothiopyranyl; benzpyrazolyl; dihydrobenzofuryl; dihydrobenzothienyl; dihydrobenzothiopyranyl; dihydrobenzothiopyranyl sulfone; dihydrobenzopyranyl; indolinyl; isochromanyl; isoindolinyl; naphthyridinyl; phthalazinyl; piperonyl; purinyl; pyridopyridyl; quinazolinyl; tetrahydroquinolinyl; thienofuryl; thienopyridyl; thienothienyl and the like.
The term xe2x80x9cheteroarylxe2x80x9d refers to an aromatic heterocycle.
xe2x80x9cSubstituted heterocycloxe2x80x9d or xe2x80x9csubstituted heteroarylxe2x80x9d refer to the respective moiety (heterocyclo or heteroaryl) substituted with one or more substituents. Exemplary substituents include one or more alkyl groups or one or more groups described as alkyl substituents. Substituted heterocyclo or heteroaryl may be substituted with a mono-oxo to give for example 4-oxo-1H-quinoline. Substituted heterocyclo or heteroaryl may also be substituted with a substituted aryl or a second substituted heterocyclo to give for example a 4-phenylimidazol-1-yl or a 4-(pyridin-3-yl)-imidazol-1-yl.
The term xe2x80x9chydroxy protecting groupxe2x80x9d refers to groups known in the art for such purpose. Commonly used hydroxy protecting groups are disclosed, for example, in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley and Sons, New York (1991), which is incorporated herein by reference. Illustrative hydroxyl protecting groups include but not limited to tetrahydropyranyl; benzyl; methylthiomethyl; ethythiomethyl; pivaloyl; phenylsulfonyl; triphenylmethyl; trisubstituted silyl such as trimethyl silyl, triethylsilyl, tributylsilyl, tri-isopropylsilyl, t-butyldimethylsilyl, tri-t-butylsilyl, methyldiphenylsilyl, ethyldiphenylsilyl, t-butyldiphenylsilyl and the like; acyl and aroyl such as acetyl, pivaloylbenzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryl and the like.
In addition to the explicit substitutions at the above-described groups, the inventive compounds may include other substitutions where applicable. For example, the erythromycin backbone or backbone substituents may be additionally substituted (e.g., by replacing one of the hydrogens or by derivatizing a non-hydrogen group) with one or more substituents such as C1-C5 alkyl, C1-C5 alkoxy, phenyl, or a functional group. Illustrative examples of suitable functional groups include but are not limited to alcohol, sulfonic acid, phosphine, phosphonate, phosphonic acid, thiol, ketone, aldehyde, ester, ether, amine, quaternary ammonium, imine, amide, imide, imido, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, carbamate, acetal, ketal, boronate, cyanohydrin, hydrazone, oxime, hydrazide, enamine, sulfone, sulfide, sulfenyl, and halogen.
Preferred embodiments of compounds of the present invention includes compounds of the formula I 
wherein:
X is hydrogen or fluoride;
R2 is hydrogen, xe2x80x94COCH3 or xe2x80x94COPhenyl;
R13 is methyl, propyl, vinyl, butyl, 3-butenyl, 3-hydroxylbutyl, 2-fluoroethyl or 2-azidoethyl;
R6 is xe2x80x94ORa wherein Ra is hydrogen, C1-C5 alkyl, or xe2x80x94YZ
wherein Y is a C1-C10 alkyl, C2-C10 alkenyl, or C2-C10 alkynyl, more preferably C3-C6 alkyl, C3-C6 alkenyl or C3-C6 alkynyl; and, Z is a substituted aryl, unsubstituted aryl, substituted heterocyclo or unsubstituted heterocyclo, more preferably a substituted or unsubstituted heteroaryl;
R is hydrogen or Ra.
Illustrative examples of preferred substituted and unsubstituted heterocyles for R6 or R include but are not limited to 
wherein the substituted or unsubstituted heteroaryl or its tautomeric forms may be attached at any suitable atom.
Additional examples of substituted or unsubstituted heterocycles for R6 or R include nucleic acid bases and derivatives thereof such as 
wherein the nucleic acid base or derivative may be attached at any suitable atom.
Particularly preferred compounds of the present invention include: 
wherein
X is H or F;
R13 is methyl, propyl, or vinyl; and,
Ra is selected from the group consisting of 
Especially preferred compounds of the present invention include those of formula III 
wherein X is hydrogen or fluoride and Ra is selected from the group consisting of 
Particularly preferred are compounds of Formula I wherein:
X is hydrogen or fluoride;
R is hydrogen;
R2 is hydrogen, xe2x80x94COCH3 or xe2x80x94COPhenyl;
R13 is methyl, propyl or vinyl; and,
R6 is selected from a group consisting of 3-(quinolin-3-yl)prop-2-enyl; 3-(quinolin-3-yl)prop-2-ynyl; 3-(quinolin-6-yl)prop-2-enyl; 3-(quinolin-6-yl)prop-2-ynyl; 3-(quinolin-7-yl)prop-2-enyl; 3-phenylprop-2-enyl; 3-(naphth-1-yl)prop-2-enyl; 3-(naphth-1-yl)prop-2-ynyl; 3-(naphth-2-yl)prop-2-ynyl; 5-phenylpent-4-en-2-ynyl; 3-(fur-2-yl)prop-2-ynyl; 3-(thien-2-yl)prop-2-enyl; 3-(carbazol-3-yl)prop-2-enyl; and 3-(quinoxalin-6-yl)prop-2-enyl.
Particularly preferred groups for R include H, phenyl, C1-C8-alkyl or C1-C8-alkenyl optionally substituted with one or more substituents selected from the group of phenyl, hydroxy, and the following substituted heterocyclo groups. 
Aglycone intermediates may be prepared by methods described in U.S. Pat. Nos. 5,672,491; 5,830,750; 5,843,718; 5,712,146; 5,962,290; 6,022,731; 6,066,721; 6,077,696; and, 6,080,555 which are all incorporated herein by reference. In one embodiment, xe2x80x9cunnaturalxe2x80x9d erythromycin precursor may be prepared by a method in which an appropriate thioester diketide substrate is provided to a 6-deoxyerythronolide B synthase (xe2x80x9cDEBSxe2x80x9d) that is unable to act on its natural substrate, propionyl CoA, due to a mutation in the ketosynthase domain of module 1 of DEBS. This recombinant DEBS can be expressed in the natural host that normally produces erythromycin, Saccharopolyspora erythraea, or the entire PKS gene cluster can be inserted by plasmid in a suitable host such as S. coelicolor (see e.g., Jacobsen et al, Science 277: 367-369 (1997)) or S. lividans which has been modified to delete its endogenous actinorhodin polyketide synthesis mechanism. For example, a suitable host would be S. coelicolor CH999/pJRJ2, which expresses a mutant 6-DEB synthase having an inactivated module 1 ketosynthase.
A cell free system as described in U.S. Pat. No. 6,080,555 and PCT Publication No. WO 97/02358 may also be employed by producing the relevant PKS proteins recombinantly and effecting their secretion or lysing the cells containing them. A typical cell-free system would include the appropriate PKS, NADPH and an appropriate buffer and substrates required for the catalytic synthesis of polyketides.
Further, the appropriate thioester diketide substrates can be provided to PKS enzymes other than the 6-DEB synthase of Saccharopolyspora erythraea. Other PKS enzymes include the 6-DEB synthase of Micromonospora megalomicea and its KS1xc2x0 derivative described in U.S. Ser. Nos. 60/190,024 and 60/158,305, the oleandolide PKS and its KS1xc2x0 derivative described in PCT Application No. US 99/24478, and the narbonolide PKS and its KS1xc2x0 derivative described in PCT Publication No. WO 99/61599, all of which are incorporated herein by reference.
For those aglycone intermediates wherein R13 is methyl, diketide feeding is not required because the desired aglycone may be produced by the recombinant host cell Streptomyces coelicolor CH999/pCK7, as further described herein.
The aglycones thus prepared are then added to the fermnentation broth of Saccharopolyspora erythraea strains which glycosylate at the C-3 and C-5 positions, hydroxylate at C-12, and optionally hydroxylate at the C-6 position, depending on the strain employed. Preferred embodiments of the hydroxylations and glycosylations are compounds of the general formula 
wherein R13 is as described previously. These and other xe2x80x9cunnaturalxe2x80x9d erythromycin compounds detailed above are used as starting materials for further chemical synthesis.
The biosynthetically derived starting material is further modified by chemical synthesis. The subsequent modifications include halogenation at C-2; formation of the keto group at C-3; formation of a cyclic carbamate at C-11 and C-12; derivation at the C-6 hydroxyl (where a hydroxyl exists at this position); and combinations thereof. All resulting compounds (including all intermnediates) are considered part of the present invention.
When a hydroxyl exists at the C-6 position of the biologically derived starting material, it is modified typically with an alkyl group. Scheme 1 illustrates one method for alkylating the C-6 hydroxyl starting from the biologically derived C-13 modified erythromycin A. 
Briefly, the C-9 keto group of the starting erythromycin compound 1 is protected with a keto protecting group, preferably by converting the keto group into a derivatized oxime (xe2x95x90NORxe2x80x2 wherein Rxe2x80x2 is a substituted or unsubstituted moiety such as C1-C12 alkyl, C3-C12 cycloalkyl, C6-C10 aryl and heteroaryl. A preferred derivatized oxime is of the formula xe2x95x90NORxe2x80x2 wherein Rxe2x80x2 is isopropoxycyclohexyl as in compound 3. Alternatively, instead of forming the oxime, the C-9 keto group may be reduced to a hydroxyl which may be optionally protected with a selective hydroxyl protecting group prior to the alkylation reaction at the C-6 hydroxyl.
The sugar hydroxyls (2xe2x80x2 and the 4xe2x80x3 positions) are protected using reagents such as acetic anhydride, benzoic anhydride, benzyl chloroformate, hexamethyldisilazane, or a trialkylsilyl chloride in an aprotic solvent. Illustrative examples of aprotic solvents include dichloromethane, chloroform, tetrahydrofuran, N-methyl pyrrolidone, dimethyl sulfoxide (xe2x80x9cDMSOxe2x80x9d), dimethyl formamide (xe2x80x9cDMFxe2x80x9d) and trimethylsilylimidazole. Preferred protecting agents include trimethylsilyl chloride in trimethylsilylimidazole.
The resulting compound 4 is reacted with an alkylating agent such as alkyl halides, sulfonates and tosylates, in the presence of a base to form compound 5. Preferred alkylating agents include alkylbromide RaBr such as methyl bromide, allyl bromide, propargyl bromide, 2-fluoroethyl bromide, cinnamyl bromide, and crotonyl bromide. Suitable bases include potassium hydroxide, sodium hydride, potassium isopropoxide, potassium t-butoxide, and an aprotic solvent.
Once the alkylation of the C-6-hydroxyl is completed, the sugar residues and the macrolide ring may be deprotected. Deprotection of the glycoside moieties is conducted as described by T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, infra. Similar conditions result in converting the derivatized oxime to xe2x95x90NOH. If formation of the underivatized oxime is not concurrent with deprotection, the conversion to the oxime is conducted separately.
The oxime is removed and converted to a keto group by standard methods known in the art. Deoximating agents include inorganic sulfur oxide compounds such as sodium hydrogen sulfite, sodium pyrosulfate, sodium thiosulfate, and the like. In this case, protic solvents are used, such as water, methanol, ethanol, isopropanol, trimethyl silanol, and mixtures thereof. In general, the deoximation reaction is conducted in the presence of an organic acid such as formic acid. The product of the deoximation reaction is compound 7, the C-6 alkylated derivative of the starting material.
The C-6 alkylated compound 7 may be further modified. For example, when Ra is allyl, it may be treated with osmium tetroxide to provide the 2,3-dihydroxylpropyl compound, which can further be esterified at each oxygen atom. The 6-O-allyl compound may also be oxidized with m-chloroperoxybenzoic acid in an aprotic solvent to provide the epoxy compound which can be opened with amines or N-containing heteroaryl compounds. Alternatively the allyl side chain may be oxidized under Wacker conditions to provide the substituent xe2x80x94Oxe2x80x94CH2C(xe2x95x90O)CH3, or ozonized to provide the aldehyde. The aldehyde can then be converted to the oxime which in turn can be reacted with a dehydration agent in an aprotic solvent to yield a nitrile. Alternatively, the aldehyde can be reacted with a suitable amine and reduced in the presence of a borohydride reducing agent to provide an amine.
A preferred modification of compound 7 is the formation of a keto group at C-3 as illustrated by Scheme 2. 
The C-3 sugar is removed with an acid, preferably aqueous HCl, or a deglycosylating enzyme to yield the corresponding des-cladinose derivative 8. Suitable acids include hydrochloric, sulfuric, chloroacetic, trifluoroacetic and the like in the presence of alcohol and water. Reaction times are typically 0.5-24 hours at a temperature of approximately between xe2x88x9210 and 35xc2x0 C. The free hydroxyl of the remaining sugar moiety (xe2x80x9cdesoaminexe2x80x9d) at the C-5 position of the erythromycin backbone is selectively protected with a protecting group such as acetic or benzoic anhydride (whereby R2 is Ac or Bz). The C-3 hydroxyl is oxidized to a keto group to yield compound 10. In this procedure, an oxidizing agent such as N-chlorosuccinimide-dimethyl sulfide or a carbodiimide-dimethylsufoxide is used. Typically, compound 9 is added to a pre-formed N-chlorosuccinimide and dimethyl sulfide complex in a chlorinated solvent such as methylene chloride at xe2x88x9210-25xc2x0 C. After being stirred for 0.5 to 4 hours, a tertiary amine such as triethylamine is added to produce the corresponding ketone.
Compound 10 may also be further modified preferably with a cyclic carbamate at C-11 and C-12 positions. One method for forming the carbamate moiety is outlined by Scheme 3. 
Briefly, compound 11 is prepared from compound 10, for example, in a two-step procedure. First, the C-11 hydroxyl group is preferentially converted to a leaving group by reaction with an alkyl or arylsulfonyl chloride, such as methanesulfonyl chloride, in the presence of an organic base, like pyridine. In the next step, the leaving group is eliminated by treatment with diazabicycloundecane in a suitable solvent like acetone to afford the double bond between C-10 and C-11. Compound 11 is reacted with 1,1xe2x80x2-carbonyldiimidazole and then an amine RNH2. Removal of the 2xe2x80x2-hydroxyl protecting group is effected with methanol to yield compound 13. Alternatively, compound 10 may be reacted with 1,1xe2x80x2-carbonyldiimidazole in the presence of a base, like sodium hydride, to yield compound 12 directly which may then be reacted with RNH2 to prepare the desired product, compound 13.
Preferred embodiments of the inventive compounds generally include a substituted aryl or heterocyclo at R or Ra. For those compounds wherein R is a substituted aryl or heterocyclo, Ra is preferably C1-C10 alkyl, and more preferably C1-C5 alkyl with CH3 being the most preferred. To obtain compounds where R is a substituted aryl or heterocyclo, the corresponding amine, RNH2, is used as described by Scheme 3. Illustrative examples of suitable xe2x80x94R groups include but are not limited to: quinolin-4ylbutyl; 4-phenylimidazol-1-ylbutyl; 4-(pyridin-3-yl)imidazol-1-ylbutyl; 4-(pyridin-3-yl-imidazol-1-ylbutyl; pyridin-4-ylbutyl; 3H-imidazo[4,5-b]pyridin-3-ylbutyl; 1H-imidazo[4,5-b]pyridin-1-ylbutyl; 1H-imidazo[4,5-c]pyridin-1-ylbutyl; 3H-imidazo[4,5-c]pyridin-3-ylbutyl; 1H-imidazo[4,5-c]pyridin-1-ylbutyl; purin-7-ylbutyl; purin-9-ylbutyl; and 1H-imidazo[4,5-b]pyridin-1-ylbut-2-enyl; and, 4-(pyrimidin-5-yl)imidazol-1-ylbutyl.
For those compounds wherein Ra is substituted aryl or heterocyclo, R is preferably hydrogen. Although these compounds may be made by any suitable method, a two step modification at the C-6 hydroxyl is preferred. In general, the C-6 hydroxyl is modified as previously described in Scheme 1 except that alkylbromide YBr wherein Y is C2-C10 alkenyl, or C2-C10 alkynyl, more preferably C3-C6 alkenyl or C3-C6 alkynyl, is used in the initial alkylation reaction at the C-6 hydroxyl. The resulting product is further modified as described by Schemes 2 and 3 to result in compound 13a. 
As shown by Scheme 4, compound 13a is reacted with a Z-halide under Heck conditions (Pd(II) or Pd(0), phosphine and amine or inorganic base) to provide compound 14 whereby Z is coupled to Y. In these compounds, the group xe2x80x94YZ together is Ra. Illustrative examples of xe2x80x94YZ include but are not limited to: 3-(quinolin-3-yl)prop-2-enyl; 3-(quinolin-3-yl)prop-2-ynyl; 3-(quinolin-6-yl)prop-2-enyl; 3-(quinolin-6-yl)prop-2-ynyl; 3-(quinolin-7-yl)prop-2-enyl; 3-phenylprop-2-enyl; 3-(naphth-1-yl)prop-2-enyl; 3-(naphth-1-yl)prop-2-ynyl; 3-(naphth-2-yl)prop-2-ynyl; 5-phenylpent-4-en-2-ynyl; 3-(fur-2-yl)prop-2-ynyl; 3-(thien-2-yl)prop-2-enyl; 3-(carbazol-3-yl)prop-2-enyl; and 3-(quinoxalin-6-yl)prop-2-enyl. These derivatives may be optionally reduced, for example, with hydrogen and palladium on carbon, to provide the corresponding compounds wherein the one or more double or triple carbon-carbon bonds in Y becomes fully saturated (e.g., propenyl to propyl).
For those compounds where R6 is hydrogen (instead of ORa), the preferred method for making C-3 keto derivatives and the C-11, 12 cyclic carbamate derivatives is described by Scheme 5. 
In these compounds, the double bond between carbons 10 and 11 is formed to yield compound 15 prior to the formation of the keto group at C-3. The resulting keto compound may be optionally halogenated at this point or the keto compound may be reacted with 1,1-carbonyldimidazole to make compound 12b. Reaction of compound 12b with an amine RNH2, followed by removal of the protecting group on the desosamine sugar results in the cyclic carbamate derivative compound 13b.
This modified protocol is also preferred when making C-3 keto derivatives and the C-11, 12 cyclic carbamate derivatives where R13 is vinyl. For these compounds, the initial alkylation at the C-6 hydroxyl (preferably to yield xe2x80x94OCH3 at this position) is accomplished as described by Scheme 1. The C-3 keto and the C-11,12 cyclic carbamate derivatives are then prepared as described by Scheme 5.
All of the end-compounds that result from reactions described by Schemes 1-5 may be optionally halogenated at C-2 to provide the corresponding halogenated counterparts. Preferred methods include treating the desired compound with a base and an electrophilic halogenating reagent such as pyridinium perbromide or N-fluorobenzenesulfonimide. Halogenated counterparts of compounds 13 and 14 may be formed by halogenating the respective compound or by halogenating its respective precursor, compound 11, prior to the formation of the cyclic carbamate. If the desired compounds are halogenated counterparts of compound 14, it is preferred to halogenate compound 14 instead of halogenating compound 11.
This invention further provides a method of treating bacterial infections, or enhancing the activity of other anti-bacterial agents, in warm-blooded animals, which comprises administering to the animals a compound of the invention alone or in admixture with a diluent or in the form of a medicament according to the invention.
When the compounds are employed for the above utility, they may be combined with one or more pharmaceutically acceptable carriers, e.g., solvents, diluents, and the like, and may be administered orally in such forms as tablets, capsules, dispersible powders, granules, or suspensions containing for example, from about 0.5% to 5% of suspending agent, syrups containing, for example, from about 10% to 50% of sugar, and elixers containing, for example, from about 20% to 50% ethanol, and the like, or parenterally in the form of sterile injectable solutions or suspensions containing from about 0.5% to 5% suspending agent in an isotnoic medium. These pharmaceutical preparations may contain, for example, from about 0.5% up to about 90% of the active ingredient in combination with the carrier, more usually between 5% and 60% by weight.
Compositions for topical application may take the form of liquids, creams or gels, containing a therapeutically effective concentration of a compound of the invention admixed with a dernatologically acceptable carrier.
In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils, as are appropriate to the nature of the active ingredient and the particular form of administration desired. Adjuvants customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA.
The preferred pharmaceutical compositions from the standpoint of ease of preparation and administration are solid compositions, particularly tablets and hard-filled or liquid-filled capsules. Oral administration of the compounds is preferred.
These active compounds may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl-cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a daily dosage of from about 0.1 mg/kg to about 400 mg/kg of animal body weight, preferably given once a day, or in divided doses two to four times a day, or in sustained release form. For most large mammals, the total daily dosage is from about 0.07 g to 7.0 g, preferably from about 100 mg to 1000 mg. Dosage forms suitable for internal use comprise from about 100 mg to 500 mg of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regiment may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The production of the above-mentioned pharmaceutical compositions and medicaments is carried out by any method known in the art, for example, by mixing the active ingredient(s) with the diluent(s) to form a pharmaceutical composition (e.g., a granulate) and then forming the composition into the medicament (e.g., tablets).
The compounds of the invention can be prepared using intermediates produced by a chemobiosynthetic procedure involving recombinant host cells and organic chemistry methodologies. Steps of this chemobiosynthetic procedure are described generally below, followed by a detailed description of each step in the enumerated Examples.
In the first general step of the method, a 6-deoxyerythronolide B (xe2x80x9c6-dEBxe2x80x9d) derivative compound is prepared by fermentation of a recombinant Streptomyces host cell. The fermentation to produce 15-methyl-6-deoxyerythronolide B and 14,15-dehydro-6-deoxyerythronolide B requires a synthetic diketide intermediate to be fed to the fermenting cells. The preparation of these synthetic diketides is described in Example 1. These synthetic diketides are substrates for a 6-deoxyerythronolide B synthase (xe2x80x9cDEBSxe2x80x9d) that is unable to act on its natural substrate (propionyl CoA) due to a mutation in the ketosynthase domain of module 1 of DEBS. This recombinant DEBS is provided by plasmid pJRJ2 in Streptomyces coelicolor CH999. S. coelicolor CH999 is described in U.S. Pat. No. 5,672,491, incorporated herein by reference. A derivative of S. coelicolor CH999, S. coelicolor K39-02, that has been genetically modified to include a ptpA gene, is described in U.S. patent application Ser. No. 09/181,833, incorporated herein by reference, can also be employed for this purpose. Plasmid pJRJ2 encodes the eryAI, eryAII, and eryAIII genes; the eryAI gene contained in the plasmid contains the KS1 null mutation. The KS1 null mutation prevents formation of the 6-deoxyerythronolide B produced by the wild-type gene unless exogenous substrate is provided. Plasmid pJRJ2 and a process for using the plasmid to prepare novel C-13-substituted erythromycins are described in PCT publication Nos. 99/03986 and 97/02358; in U.S. Pat. Nos. 6,080,555 and 6,066,721; and in U.S. patent application Ser. No. 09/311,756, filed May 14, 1999, each of which is incorporated herein by reference. The exogenous substrates provided can be prepared by the methods and include the compounds described in PCT patent application No. PCT/US00/02397 and U.S. patent application Ser. No. 09/492,733, both filed Jan. 27, 2000, by inventors G. Ashley et al., both of which are incorporated herein by reference. PKS genes other than the ery genes can also be employed; suitable genes include the KS 1 null mutation containing oleandolide and megalomicin PKS genes described in U.S. patent application Ser. Nos. 09/, filed Oct. 4, 2000 entitled Recombinant Megalomicin Biosynthetic Genes by inventors Robert McDaniel and Yana Volchegursky; 60/158,305, filed Oct. 8, 1999; and 09/428,517, filed Oct. 28, 1999, and PCT Application No. US99/24478, filed Oct. 22, 1999, each of which is incorporated herein by reference.
The fermentation to produce 14-nor-6-deoxyerythronolide B does not require diketide feeding, because the desired compound is produced by the recombinant host cell Streptornyces coelicolor CH999/pCK7. Plasmid pCK7 is described in U.S. Pat. No. 5,672,491 and comprises the DEBS genes. A derivative of plasmid pCK7, pKOS011-26, can also be used. The host cell comprising pKOS011-26 and a recombinant ptpA gene is called S. coelicolor 27-26/pKOS011-26. These host cells produce both 6-deoxyerythronolide B and 14-nor-6-deoxyerythronolide, due to the incorporation of propionyl CoA and acetyl CoA, both of which serve as substrates for DEBS.
The fermentation of Streptomyces coelicolor CH999/pJRJ2 and S. coelicolor CH999/pCK7 is described in Example 2. The isolation of the 6-deoxyerythronolide products resulting from this fermentation is also described in Example 2.
The isolated products are then added to the fermentation broth of Saccharopolyspora erythraea strains to make other useful intermediate compounds of the invention. The S. erythraea strains catalyze the biosynthesis and attachment of sugar residues to the C-3 and C-5 positions of the 6-dEB derivative compounds. These strains also comprise a functional eryK gene product and so hydroxylate the 6-dEB derivative compounds at the C-12 position. The strains differ in regard to whether a functional eryF gene product is produced. If so, then the compounds produced are hydroxylated at the C-6 position as well. If not, then a 6-deoxyerythromycin A derivative is produced. These S. erythraea fermentations are described in Example 3, together with the isolation of the erythromycin A derivative compounds from the fermentation broth.
The isolated products are then used as starting materials in the chemical synthesis of the inventive compound. For erythromycin A derivative compounds of the invention that comprise a 6-hydroxyl, Examples 4-6, 11, and 16 describe the process for alkylating the compounds to make the C-6-O-alkyl, C-6-O-allyl, and C-6-O-propargyl intermediates.
For erythromycin A derivative compounds of the invention that comprise the C-6-O-alkyl groups, Examples 7-9 describe the process for making the 10,11-anhydro compounds of the invention.
Example 10 describes the process for making the C-2-halo compounds of the invention. In particular, the compound to be halogenated is treated with a base and an electrophilic halogenating reagent such as pyridinium perbromide or N-fluorobenzenesulfonimide. Example 12 describes the process for removing the cladinose sugar from erythromycin A derivatives containing the C-6-O-allyl group and for oxidation of the resulting C-3-hydroxyl group to the ketone. Example 13 illustrates the conversion of the compounds containing the C-6-O-allyl group to several useful intermediates in the synthesis of compounds of the invention. Example 14 describes the synthesis of a compound of the Formula I wherein R=H, R2=H, X=H and R6=O-allyl. Example 15 describes the process for conversion of macrolides containing the 6-O-allyl and 11,12-cyclic carbamate functionalities to compounds of the formula I via the Heck reaction and subsequent deprotection of the desosamine sugar. Example 16 describes the alkylation of the compounds to the 6-O-propargyl intermediates and Example 17 describes the conversion of the 6-O-propargyl group to 6-O-propynyl-heteroaryl compounds of formula I.
For erythromycin A derivative compounds of the invention that do not comprise a C-6 hydroxyl, Examples 18-20 describe the process for making the 10,11-anhydro compounds of the invention. These reaction sequences are depicted in Schemes 5 and 6.
Example 21 describes the process for the synthesis of 1H-imidazo[4,5-b]pyridine-1-(4-amino-2-butene), an amine used in the synthesis of compounds of the invention wherein R=1H-imidazol[4,5-b]pyridine.
The process for conversion of the 10,11-anhydro compounds into the carbamate derivative compounds of the invention is described in Examples 22 and 23. The amines used in the synthesis of the carbamate derivative compounds of formula I are either commercially available or can be readily prepared as described in Denis et al, Bioorg. Med. Chem. Lett. 9:3075-3080 (1999).